Summary of Work: There are more than 42 million people infected by the HIV virus worldwide where 5 million new infections occurred during 2002. Although antiviral therapy can extend the life of individuals, the death toll continues to rise: 3.1 million people, the highest number since the epidemic began, died from AIDS last year. Although antiviral nucleoside analog therapy successfully delays progression of HIV infection to AIDS, these drugs cause unwelcome side effects by inducing mitochondrial toxicity. Current antiviral nucleoside analog therapy against HIV results in compromised mitochondrial function due to selective inhibition of the mitochondrial DNA polymerase. As much as 20% of patients undergoing AZT treatment develop a mitochondrial dysfunctional disease known as red ragged fiber disease and D4T and ddC cause neuropathy in 15-20% of patients. The mode and effect of antiviral nucleotide analogs, by AZT, ddI, 3TC, D4T and others on the inhibition and fidelity of the mitochondrial DNA polymerase and mitochondrial DNA replication are poorly understood. What structural properties set this polymerase apart from the nuclear DNA polymerases to give rise to its inhibition patterns is poorly understood. We previously evaluated the ability of such analogs to inhibit DNA synthesis by the human mitochondrial DNA polymerase gamma (pol gamma) by comparing the insertion and exonucleolytic removal of six antiviral nucleotide analogs. The structural elements responsible for this inhibition and the amino acids that interact with these analogs are unknown. In this last year, we explored the role of three highly conserved amino acid residues in the active site of human pol g that modulate selection of nucleotide analogs as substrates for incorporation. Sequence alignments, crystal structures and mutagenesis studies of Family A DNA polymerases led us to change Tyr951 and Tyr955 in polymerase motif B to Phe and Ala, and Glu895 in polymerase motif A was changed to Ala. The mutant polymerases were tested for their ability to incorporate natural nucleotides and the five antiviral nucleoside analogs currently approved for antiviral therapy: AZT, ddC, D4T, 3TC and carbovir. Steady state kinetic analysis of the pol g derivatives with the normal and antiviral nucleotides demonstrated that Tyr951 is largely responsible for the ability of pol g to incorporate dideoxynucleotides and D4T-MP. Mutation of Tyr951 to Phe renders the enzyme resistant to dideoxynucleotides and D4T-TP without compromising the activity of the polymerase. Alteration of Glu895 and Tyr955 to Ala had the largest effect on overall polymerase activity with normal nucleotides, producing dramatic increases in Km(dNTP) and large decreases in kcat. Mutation of Tyr955 in pol g causes the degenerative disease PEO in humans, and we showed that this residue partially accounts for the ability of pol g to incorporate D4T-MP and carbovir. Alteration of Glu895 to Ala slightly increased discrimination against dideoxynucleotides and D4T-TP. These results should form the basis for rational structure based drug design of anti-HIV nucleoside analogs with reduced mitochondrial toxicity.